Expression of CYP450 enzymes in human fetal membranes and its implications in xenobiotic metabolism during pregnancy

Abstract

Background: Environmental exposure to toxicants is a major risk factor for spontaneous preterm birth (PTB, <37 weeks). Toxicants and drugs administered to patients are metabolized primarily by the cytochrome P450 (CYP450) system. Along with the adult and fetal liver, the placenta, a critical feto-maternal interface organ, expresses CYP450 enzymes that metabolize these xenobiotics. However, the contribution of the fetal membranes, another tissue of the feto-maternal interface, to the expression of CYP450 enzymes and the detoxification of xenobiotics remains unknown.

Aims: This study characterized CYP450 expression and determined the functional activity of CYP450 enzymes in fetal membranes.

Main methods: RNA sequencing (RNA-Seq) of placental and fetal membrane tissues and cells was done. Differential expressions of CYP450 genes were compared and validated via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) between the two tissues. The functional activity of major CYP450 enzymes was determined using a fluorophore-based enzymatic assay in the presence and absence of their corresponding inhibitors.

Key findings: With the exception of genes that regulate cholesterol metabolism, the expression profile of CYP450 genes was similar between placental and fetal membranes tissues/cells. RT-qPCR analysis confirmed these findings with significant levels of mRNA for major CYP450 genes being detectable in amnion epithelial cells (AECs) and chorion trophoblasts cells (CTCs). Biochemical analyses revealed significant CYP450 enzymatic activities that were sensitive to specific inhibitors for both AECs and CTCs, suggesting that the genes were expressed as functional enzymes.

Significance: This is the first study to determine global expression of CYP450 enzymes in fetal membranes which may play a role in xenobiotic metabolism during pregnancy. Given that many women are exposed to environmental toxins or require medications during pregnancy, a better understanding of their role in metabolism is required to develop safer therapeutics and prevent adverse outcomes.

Keywords: Cytochrome P450; Drug metabolism; Fetal membrane; Pharmacokinetics; Placenta; Pregnancy; Toxicant metabolism.

Fig. 1.

CYP450 expression profiles in human placental and fetal membrane tissues. (A) Relative CYP450 expression (log₁₀ scale) in human term placental (PLA) and fetal membrane (FM) tissues (n = 5 each). Colors indicate differential expression (red: transcript levels greater than the median; black: transcript levels equal to the median; green: transcript levels below the median). (B) Volcano plots of CYP enzymes of FM in comparison with PLA. Differentially expressed genes were shown in red dots. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2.

CYP expression profile in human FM and BeWo cells. (A) Relative CYP450 expression (log₁₀ scale) in BeWo, amnion epithelial cells (AECs), amnion mesenchymal cells (AMCs), chorion trophoblast cells (CTCs) and decidual cells (DECs) (n = 3 each). Colors indicate differential expression (red: transcript levels greater than the median; black: transcript levels equal to the median; green: transcript levels below the median). (B) Venn diagram depicting significantly higher expression of CYP450 genes (FDR ≤ 0.05; log₂ fold change ≥1) in FM cells compared to BeWo. (C) Venn diagram depicting significantly lower expression of CYP450 genes (FDR ≤ 0.05; log₂ fold change ≤1) in FM cells compared to BeWo. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

mRNA expression levels of CYP enzymes in FM cells compared to BeWo cells. Relative fold gene expression (in terms of 2^−ΔΔCT) of various CYP450 enzymes (A) CYP3A4, (B) CYP2D6, (C) CYP1A2, (D) CYP2C9, (E) CYP2J2, (F) CYP2C8 and (G) CYP2E1 normalized to GAPDH. Data is presented as mean ± standard error of the mean, with n = 3 per cell type. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 or ****p ≤ 0.0001.

Fig. 4.

Functional activity of major CYP450 enzymes in human term fetal membranes. Enzymatic activities of (A) CYP3A4, (B) CYP1A2, (C) CYP2J2, (D) CYP2C8 and (E) CYP2E1 in BeWo cells, AECs, AMCs, CTCs and DECs were measured in terms of relative fluorescence units. All experiments were conducted in triplicates, and data was represented as Geometric mean. Red dotted lines represent CYP450 enzyme activity in the presence of their corresponding inhibitors, and blue dotted lines represent CYP450 enzyme activity without inhibitors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 or ****p ≤ 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

Functional activity of major CYP450 enzymes in human term fetal membranes. Enzymatic activities of (A) CYP3A4, (B) CYP1A2, (C) CYP2J2, (D) CYP2C8 and (E) CYP2E1 in BeWo cells, AECs, AMCs, CTCs and DECs were measured in terms of relative fluorescence units. All experiments were conducted in triplicates, and data was represented as Geometric mean. Red dotted lines represent CYP450 enzyme activity in the presence of their corresponding inhibitors, and blue dotted lines represent CYP450 enzyme activity without inhibitors. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 or ****p ≤ 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.

Summary of expression of major CYP450 enzymes in the human fetal membranes and the maternal decidua. Transcript levels were compared with placental trophoblasts (PT), and the enzymes were labeled as active (red) or inactive (blue) based on functional activity. AECs and CTCs act as the metabolizing layers of the fetal membranes along with the placenta. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)